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1.
J Cell Physiol ; 238(3): 631-646, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36727620

RESUMO

A common adverse response to the clinical use of glucocorticoids (GCs) is elevated intraocular pressure (IOP) which is a major risk factor for glaucoma. Elevated IOP arises due to impaired outflow of aqueous humor (AH) through the trabecular meshwork (TM). Although GC-induced changes in actin cytoskeletal dynamics, contractile characteristics, and cell adhesive interactions of TM cells are believed to influence AH outflow and IOP, the molecular mechanisms mediating changes in these cellular characteristics are poorly understood. Our studies focused on evaluating changes in the cytoskeletal and cytoskeletal-associated protein (cytoskeletome) profile of human TM cells treated with dexamethasone (Dex) using label-free mass spectrometric quantification, identified elevated levels of specific proteins known to regulate actin stress fiber formation, contraction, actin networks crosslinking, cell adhesion, and Wnt signaling, including LIMCH1, ArgBP2, CNN3, ITGBL1, CTGF, palladin, FAT1, DIAPH2, EPHA4, SIPA1L1, and GPC4. Several of these proteins colocalized with the actin cytoskeleton and underwent alterations in distribution profile in TM cells treated with Dex, and an inhibitor of Abl/Src kinases. Wnt/Planar Cell Polarity (PCP) signaling agonists-Wnt5a and 5b were detected prominently in the cytoskeletome fraction of TM cells, and studies using siRNA to suppress expression of glypican-4 (GPC4), a known modulator of the Wnt/PCP pathway revealed that GPC4 deficiency impairs Dex induced actin stress fiber formation, and activation of c-Jun N-terminal Kinase (JNK) and Rho kinase. Additionally, while Dex augmented, GPC4 deficiency suppressed the formation of actin stress fibers in TM cells in the presence of Dex and Wnt5a. Taken together, these results identify the GPC4-dependent Wnt/PCP signaling pathway as one of the crucial upstream regulators of Dex induced actin cytoskeletal reorganization and cell adhesion in TM cells, opening an opportunity to target the GPC4/Wnt/PCP pathway for treatment of ocular hypertension in glaucoma.


Assuntos
Actinas , Proteínas do Citoesqueleto , Citoesqueleto , Dexametasona , Glucocorticoides , Glipicanas , Malha Trabecular , Humanos , Actinas/metabolismo , Células Cultivadas , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Dexametasona/farmacologia , Glaucoma/metabolismo , Glaucoma/patologia , Glucocorticoides/farmacologia , Glipicanas/deficiência , Glipicanas/metabolismo , Pressão Intraocular , Malha Trabecular/citologia , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Citoesqueleto/metabolismo , Polaridade Celular/efeitos dos fármacos , Quinases Associadas a rho/metabolismo , Fibras de Estresse/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos
2.
J Med Chem ; 64(21): 15639-15650, 2021 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-34590489

RESUMO

Hepatocellular carcinoma (HCC) is rising steadily in incidence, and more effective methods are needed for early detection and image-guided surgery. Glypican-3 (GPC3) is a cell surface biomarker that is overexpressed in early-stage cancer but not in cirrhosis. An IRDye800-labeled 12-mer amino acid sequence was identified, and specific binding to GPC3 was validated in vitro and in orthotopically implanted HCC tumors in vivo. Over 4-fold greater binding affinity and 2-fold faster kinetics were measured by comparison with previous GPC3 peptides. Photoacoustic images showed peak tumor uptake at 1.5 h post-injection and clearance within ∼24 h. Laparoscopic and whole-body fluorescence images showed strong intensity from tumor versus adjacent liver with about a 2-fold increase. Immunofluorescence staining of human liver specimens demonstrated specific binding to HCC versus cirrhosis with 79% sensitivity and 79% specificity, and normal liver with 81% sensitivity and 84% specificity. The near-infrared peptide is promising for early HCC detection in clinical trials.


Assuntos
Carcinoma Hepatocelular/genética , Glipicanas/genética , Neoplasias Hepáticas/genética , Animais , Carcinoma Hepatocelular/diagnóstico por imagem , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Glipicanas/deficiência , Humanos , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas Experimentais/diagnóstico por imagem , Neoplasias Hepáticas Experimentais/genética , Camundongos , Camundongos Nus , Estrutura Molecular , Imagem Óptica , Técnicas Fotoacústicas , Relação Estrutura-Atividade
3.
Stem Cells ; 37(7): 899-909, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30977233

RESUMO

As a morphogen, Sonic Hedgehog (Shh) mediates signaling at a distance from its sites of synthesis. After secretion, Shh must traverse a distance through the extracellular matrix (ECM) to reach the target cells and activate the Hh response. ECM proteins, in particular, the heparan sulfate proteoglycans (HSPGs) of the glypican family, have both negative and positive effects on Shh signaling, all attributed to their ability to bind Shh. Using mouse embryonic stem cell-derived mosaic tissues with compartments that lack the glycosyltransferases Exostosin1 and Exostosin2, or the HSPG core protein Glypican5, we show that Shh accumulates around its source cells when they are surrounded by cells that have a mutated ECM. This accumulation of Shh is correlated with an increased noncell autonomous Shh response. Our results support a model in which Shh presented on the cell surface accumulates at or near ECM that lacks HSPGs, possibly due to the absence of these Shh sequestering molecules. Stem Cells 2019;37:899-909.


Assuntos
Glipicanas/genética , Proteínas Hedgehog/genética , Células-Tronco Embrionárias Murinas/metabolismo , N-Acetilglucosaminiltransferases/genética , Transdução de Sinais/genética , Animais , Diferenciação Celular , Linhagem da Célula/genética , Movimento Celular , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Edição de Genes/métodos , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Glipicanas/deficiência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , N-Acetilglucosaminiltransferases/deficiência , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Transporte Proteico , Transfecção , Proteína Vermelha Fluorescente
4.
Eur J Pharmacol ; 854: 39-47, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-30951720

RESUMO

Accumulating evidence has suggested that Glypican-5 (GPC5) is a tumor suppressor gene in many types of cancers. However, whether GPC5 is involved in glioma remains unknown. This study was designed to explore the expression, biological function and regulatory mechanism of GPC5 in glioma. Our results demonstrated that GPC5 expression was significantly decreased in multiple glioma cell lines. Gain-of-function experiments showed that the ectopic expression of GPC5 markedly inhibited the proliferation, invasion and Wnt/ß-catenin signaling of glioma cell lines. GPC5 was identified as a target gene of microRNA-301b (miR-301b). Further data showed that miR-301b expression was significantly up-regulated in glioma tissues and cell lines. In addition, miR-301b expression was inversely correlated with GPC5 expression in clinical glioma tissues. The overexpression of miR-301b promoted the proliferation, invasion and Wnt/ß-catenin signaling of glioma cell lines, whereas the inhibition of miR-301b showed the opposite effect. However, the silencing of GPC5 significantly reversed the antitumor effect of miR-301b inhibition. Overall, our results revealed a tumor suppressive role of GPC5 in glioma and suggested that GPC5 expression was regulated by miR-301b. Our study indicates that the inhibition of miR-301b represses the proliferation and invasion of glioma cells by up-regulating GPC5 expression.


Assuntos
Glioma/patologia , Glipicanas/genética , MicroRNAs/genética , Via de Sinalização Wnt/genética , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glipicanas/deficiência , Humanos , Invasividade Neoplásica
5.
Nat Genet ; 49(10): 1468-1475, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28869591

RESUMO

Osteoporosis is a common disease diagnosed primarily by measurement of bone mineral density (BMD). We undertook a genome-wide association study (GWAS) in 142,487 individuals from the UK Biobank to identify loci associated with BMD as estimated by quantitative ultrasound of the heel. We identified 307 conditionally independent single-nucleotide polymorphisms (SNPs) that attained genome-wide significance at 203 loci, explaining approximately 12% of the phenotypic variance. These included 153 previously unreported loci, and several rare variants with large effect sizes. To investigate the underlying mechanisms, we undertook (1) bioinformatic, functional genomic annotation and human osteoblast expression studies; (2) gene-function prediction; (3) skeletal phenotyping of 120 knockout mice with deletions of genes adjacent to lead independent SNPs; and (4) analysis of gene expression in mouse osteoblasts, osteocytes and osteoclasts. The results implicate GPC6 as a novel determinant of BMD, and also identify abnormal skeletal phenotypes in knockout mice associated with a further 100 prioritized genes.


Assuntos
Densidade Óssea/genética , Calcâneo/patologia , Estudo de Associação Genômica Ampla , Osteoporose/genética , Polimorfismo de Nucleotídeo Único , Animais , Modelos Animais de Doenças , Feminino , Fêmur/química , Perfilação da Expressão Gênica , Glipicanas/deficiência , Glipicanas/genética , Glipicanas/fisiologia , Transtornos do Crescimento/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Anotação de Sequência Molecular , Osteoblastos/metabolismo , Osteocondrodisplasias/congênito , Osteocondrodisplasias/genética , Osteoclastos/metabolismo , Osteócitos/metabolismo , Osteoporose/patologia , Fenótipo
6.
J Cell Biol ; 216(9): 2911-2926, 2017 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-28696225

RESUMO

Autosomal-recessive omodysplasia (OMOD1) is a genetic condition characterized by short stature, shortened limbs, and facial dysmorphism. OMOD1 is caused by loss-of-function mutations of glypican 6 (GPC6). In this study, we show that GPC6-null embryos display most of the abnormalities found in OMOD1 patients and that Hedgehog (Hh) signaling is significantly reduced in the long bones of these embryos. The Hh-stimulatory activity of GPC6 was also observed in cultured cells, where this GPC increased the binding of Hh to Patched 1 (Ptc1). Consistent with this, GPC6 interacts with Hh through its core protein and with Ptc1 through its glycosaminoglycan chains. Hh signaling is triggered at the primary cilium. In the absence of Hh, we observed that GPC6 is localized outside of the cilium but moves into the cilium upon the addition of Hh. We conclude that GPC6 stimulates Hh signaling by binding to Hh and Ptc1 at the cilium and increasing the interaction of the receptor and ligand.


Assuntos
Fêmur/metabolismo , Glipicanas/metabolismo , Transtornos do Crescimento/metabolismo , Proteínas Hedgehog/metabolismo , Osteocondrodisplasias/congênito , Osteogênese , Tíbia/metabolismo , Animais , Proliferação de Células , Cílios/metabolismo , Modelos Animais de Doenças , Fêmur/embriologia , Predisposição Genética para Doença , Glicosaminoglicanos/metabolismo , Glipicanas/deficiência , Glipicanas/genética , Transtornos do Crescimento/embriologia , Transtornos do Crescimento/genética , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Osteocondrodisplasias/embriologia , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Receptor Patched-1/metabolismo , Fenótipo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Tíbia/embriologia , Fatores de Tempo , Transfecção , Proteína GLI1 em Dedos de Zinco/metabolismo
7.
Oncotarget ; 7(28): 44406-44416, 2016 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-27259271

RESUMO

Gastric cancer is a prevalent tumor that is usually detected at an advanced metastatic stage. Currently, standard therapies are mostly ineffective. Here, we report that Glypican-3 (GPC3) is absent in invasive tumors and metastatic lymph nodes, in particular in aggressive and highly disseminated signet ring cell carcinomas. We demonstrate that loss of GPC3 correlates with poor overall survival in patients. Moreover, we show that absence of GPC3 causes up-regulation of MAPK/FoxM1 signaling and that blockade of this pathway alters cellular invasion. An inverse correlation between GPC3 and FoxM1 is also shown in patient samples. These data identify GPC3 as a potential metastasis suppressor gene and suggest its value as a prognostic marker in gastric cancer. Development of therapies targeting signaling downstream of GPC3 are warranted.


Assuntos
Glipicanas/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Feminino , Genes Supressores de Tumor , Glipicanas/deficiência , Glipicanas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias Gástricas/metabolismo
8.
Mech Dev ; 138 Pt 3: 279-90, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26459057

RESUMO

The Wnt/Planar Cell Polarity (PCP) pathway controls cell morphology and behavior during animal development. Several zebrafish mutants were identified as having perturbed Wnt/PCP signaling. Many of these mutants have defects in craniofacial formation. To better understand the role that Wnt/PCP plays in craniofacial development we set out to identify which of the mutants, known to be associated with the Wnt/PCP pathway, perturb head cartilage formation by disrupting chondrocyte morphology. Here we demonstrate that while vang-like 2 (vangl2), wnt11 and scribbled (scrib) mutants have severe craniofacial morphogenesis defects they do not display the chondrocyte stacking and intercalation problems seen in glypican 4 (gpc4) and wnt5b mutants. The function of Gpc4 or Wnt5b appears to be important for chondrocyte organization, as the neural crest in both mutants is specified, undergoes migration, and differentiates into the same number of cells to compose the craniofacial cartilage elements. We demonstrate that Gpc4 activity is required cell autonomously in the chondrocytes and that the phenotype of single heterozygous mutants is slightly enhanced in embryos double heterozygous for wnt5b and gpc4. This data suggests a novel mechanism for Wnt5b and Gpc4 regulation of chondrocyte behavior that is independent of the core Wnt/PCP molecules and differs from their collaborative action of controlling cell movements during gastrulation.


Assuntos
Condrócitos/metabolismo , Condrogênese/genética , Glipicanas/genética , Proteínas Wnt/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Região Branquial/embriologia , Região Branquial/metabolismo , Contagem de Células , Movimento Celular/genética , Tamanho Celular , Condrócitos/citologia , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Glipicanas/deficiência , Mutação , Crista Neural/embriologia , Crista Neural/metabolismo , Fenótipo , Proteínas Wnt/deficiência , Via de Sinalização Wnt/genética , Proteína Wnt-5a , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/deficiência
9.
Nature ; 486(7403): 410-4, 2012 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-22722203

RESUMO

In the developing central nervous system (CNS), the control of synapse number and function is critical to the formation of neural circuits. We previously demonstrated that astrocyte-secreted factors powerfully induce the formation of functional excitatory synapses between CNS neurons. Astrocyte-secreted thrombospondins induce the formation of structural synapses, but these synapses are postsynaptically silent. Here we use biochemical fractionation of astrocyte-conditioned medium to identify glypican 4 (Gpc4) and glypican 6 (Gpc6) as astrocyte-secreted signals sufficient to induce functional synapses between purified retinal ganglion cell neurons, and show that depletion of these molecules from astrocyte-conditioned medium significantly reduces its ability to induce postsynaptic activity. Application of Gpc4 to purified neurons is sufficient to increase the frequency and amplitude of glutamatergic synaptic events. This is achieved by increasing the surface level and clustering, but not overall cellular protein level, of the GluA1 subunit of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) glutamate receptor (AMPAR). Gpc4 and Gpc6 are expressed by astrocytes in vivo in the developing CNS, with Gpc4 expression enriched in the hippocampus and Gpc6 enriched in the cerebellum. Finally, we demonstrate that Gpc4-deficient mice have defective synapse formation, with decreased amplitude of excitatory synaptic currents in the developing hippocampus and reduced recruitment of AMPARs to synapses. These data identify glypicans as a family of novel astrocyte-derived molecules that are necessary and sufficient to promote glutamate receptor clustering and receptivity and to induce the formation of postsynaptically functioning CNS synapses.


Assuntos
Astrócitos/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Glipicanas/metabolismo , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Animais , Astrócitos/citologia , Cerebelo/citologia , Cerebelo/metabolismo , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Feminino , Glipicanas/deficiência , Glipicanas/farmacologia , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/patologia
10.
Nat Genet ; 43(5): 459-63, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21441931

RESUMO

Severe proteinuria is a defining factor of nephrotic syndrome irrespective of the etiology. Investigation of congenital nephrotic syndrome has shown that dysfunction of glomerular epithelial cells (podocytes) plays a crucial role in this disease. Acquired nephrotic syndrome is also assumed to be associated with podocyte injury. Here we identify an association between variants in GPC5, encoding glypican-5, and acquired nephrotic syndrome through a genome-wide association study and replication analysis (P value under a recessive model (P(rec)) = 6.0 × 10(-11), odds ratio = 2.54). We show that GPC5 is expressed in podocytes and that the risk genotype is associated with higher expression. We further show that podocyte-specific knockdown and systemic short interfering RNA injection confers resistance to podocyte injury in mouse models of nephrosis. This study identifies GPC5 as a new susceptibility gene for nephrotic syndrome and implicates GPC5 as a promising therapeutic target for reducing podocyte vulnerability in glomerular disease.


Assuntos
Glipicanas/genética , Síndrome Nefrótica/genética , Adulto , Idoso , Animais , Sequência de Bases , Estudos de Casos e Controles , Primers do DNA/genética , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Glipicanas/antagonistas & inibidores , Glipicanas/deficiência , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Genéticos , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/patologia , Podócitos/metabolismo , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética
11.
Dev Biol ; 348(1): 107-18, 2010 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-20883685

RESUMO

Glypicans are conserved cell surface heparan sulfate proteoglycans expressed in a spatiotemporally regulated manner in many developing tissues including the nervous system. Here, we show that Glypican-1 (GPC1) is expressed by trigeminal placode cells as they ingress and contribute to trigeminal sensory neurons in the chick embryo. Either expression of full-length or truncated GPC1 in vivo causes defects in trigeminal gangliogenesis in a manner that requires heparan sulfate side chains. This leads to either abnormal placodal differentiation or organization, respectively, with near complete loss of the ophthalmic (OpV) trigeminal ganglion in the most severe cases after overexpression of full-length GPC1. Interestingly, modulating GPC1 alters levels of endogenous Wnt signaling activity in the forming trigeminal ganglion, as indicated by Wnt reporter expression. Accordingly, GPC1 overexpression phenocopies Wnt inhibition in causing loss of OpV placodal neurons. Furthermore, increased Wnt activity rescues the effects of GPC1 overexpression. Taken together, these results suggest that appropriate levels of GPC1 are essential for proper regulation of canonical Wnt signaling during differentiation and organization of trigeminal placodal cells into ganglia.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Glipicanas/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurogênese/fisiologia , Transdução de Sinais/fisiologia , Gânglio Trigeminal/embriologia , Proteínas Wnt/fisiologia , Animais , Embrião de Galinha , Glicosilfosfatidilinositóis/metabolismo , Glipicanas/deficiência , Glipicanas/genética , Heparitina Sulfato/fisiologia , Fenótipo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/fisiologia , Células Receptoras Sensoriais/citologia , Gânglio Trigeminal/ultraestrutura , beta Catenina/química , beta Catenina/fisiologia
12.
Dev Cell ; 14(5): 700-11, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18477453

RESUMO

Loss-of-function mutations in glypican-3 (GPC3), one of the six mammalian glypicans, causes the Simpson-Golabi-Behmel overgrowth syndrome (SGBS), and GPC3 null mice display developmental overgrowth. Because the Hedgehog signaling pathway positively regulates body size, we hypothesized that GPC3 acts as an inhibitor of Hedgehog activity during development. Here, we show that GPC3 null embryos display increased Hedgehog signaling and that GPC3 inhibits Hedgehog activity in cultured mouse embryonic fibroblasts. In addition, we report that GPC3 interacts with high affinity with Hedgehog but not with its receptor, Patched, and that GPC3 competes with Patched for Hedgehog binding. Furthermore, GPC3 induces Hedgehog endocytosis and degradation. Surprisingly, the heparan sulfate chains of GPC3 are not required for its interaction with Hedgehog. We conclude that GPC3 acts as a negative regulator of Hedgehog signaling during mammalian development and that the overgrowth observed in SGBS patients is, at least in part, the consequence of hyperactivation of the Hedgehog signaling pathway.


Assuntos
Ligação Competitiva , Embrião de Mamíferos/embriologia , Glipicanas/metabolismo , Proteínas Hedgehog/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Endocitose , Fibroblastos/citologia , Fibroblastos/metabolismo , Glipicanas/deficiência , Humanos , Camundongos , Receptores Patched , Ligação Proteica , Processamento de Proteína Pós-Traducional
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